Heme proteins catalyze a diversity of reactions in cellular metabolism. While the number and identity of the protein-derived axial ligands are of primary importance in determining the reactivity of the heme iron, a number of other protein effects are crucial in tuning the heme pocket for specific function. In the research proposed we intend to investigate the molecular mechanisms by which heme chemistry is controlled in vivo for three proteins, cytochrome oxidase, myeloperoxidase and hydroxylamine oxido-reductase. All three of these proteins catalyze unusual and biologically important reactions and each shows strong protein/chromophore interactions. Cytochrome oxidase links mitochondrial electron transport to oxygen transport by catalyzing the four electron reduction of dioxygen to water. It is also the locus of site III respiratory control and contributes directly to the transmembrane proton gradient by pumping protons stoichiometrically with electrons transported. In microbicidally active leucocytes, myeloperoxidase produces hypochlorous acid and hydroxylamine oxido-reductase catalyzes NH2OH oxidation in nitrogen metabolizing bacteria. In our cytochrome oxidase work, optical, EPR and static and time resolved resonance Raman spectroscopies, together with magnetic susceptibility measurements, will be used to test a model we've proposed for the proton pump, to identify intermediates in dioxygen reduction and to elucidate the molecular basis for enzyme heterogeneity. The preparation and characterization of appropriate model compounds and biochemical manipulation of the protein will be incorporated into this effort. Our work with myeloperoxidase and hydroxylamine oxido-reductase is collaborative; we plan to apply resonance Raman spectroscopy in order to determine chromophore structure and protein contacts, to characterize ligand binding modes and to determine the composition of catalytically important species.